The Influence of Cell Culture Density on the Cytotoxicity of Adipose-Derived Stem Cells Induced by L-Ascorbic Acid-2-Phosphate

Ascorbic acid-2-phosphate (A2-P) is an oxidation-resistant derivative of ascorbic acid that has been widely employed in culturing adipose-derived stem cells (ASCs) for faster expansion and cell sheet formation. While high dose ascorbic acid is known to induce cellular apoptosis via metabolic stress and genotoxic effects, potential cytotoxic effects of A2-P at high concentrations has not been explored. In this study, the relationship between ASC seeding density and A2-P-induced cytotoxicity was investigated. Spheroid-derived ASCs with smaller cellular dimensions were generated to investigate the effect of cell-cell contact on the resistance to A2-P-induced cytotoxicity. Decreased viability of ASC, fibroblast, and spheroid-derived ASC was noted at higher A2-P concentration, and it could be reverted with high seeding density. Compared to control ASCs, spheroid-derived ASCs seeded at the same density exhibited decreased viability in the A2-P-supplemented medium. The expression of antioxidant enzymes (catalase, SOD1, and SOD2) was enhanced in ASCs at higher seeding densities. However, their enhanced expression in spheroid-derived ASCs was less evident. Furthermore, we found that co-administration of catalase or N-acetylcysteine nullified the observed cytotoxicity. Collectively, A2-P can induce ASC cytotoxicity at higher concentrations, which can be prevented by seeding ASCs at high density or co-administration of another antioxidant.